Functions domains of factor IX will be defined by four overlapping approaches. Congenital defects provide dozens of distinct mutants, the structural bases of which can be correlated to clinical severity and specific levels of functional, in vitro interactions. Specific antisera,including monoclonal antibodies and polycolonal fractions, assist in localizing defects or functions. Synthetic peptides can be used to prepare specific antisera and to directly inhibit functional interactions. Finally, for regions in which synthetic peptides inadequately reflect surface structure, synthetic fragments can be produced by expression of mutated cDNAs in an appropriate vector which has been transfected into a cultured cell line. This proposal focuses upon distinct functional domains of factor IX. These are presented from the N to the C terminus. First, we will define the defect in a hemophilic IX which as abnormal calcium-binding properties. A synthetic leader peptide will be tested further for both stimulation of Gla formation in cell expression systems and with Dr. Suttie, for its structural requirements for interaction with the carboxylase system in vitro. Secondly, hemophilic defects involving the fourth exon will be characterized by analyses of their DNA and isolated proteins. A major effort will mounted to produce recombinant factor IX fragments containing the growth factor-like regions(s) in order to probe its role in hemostasis. Both locally with Dr. Heimark and with Dr. Stern in New York, peptides, antibodies and isolated abnormal IXs will be studied for IX binding to endothelial cells. Other potential roles of this region will be assessed by in vitro clotting and binding studies and animal survival measurements of the recombinant fragments. We will confirm that a specific antibody fraction to an exon IV eptiope recognizes a non-Gla, calcium binding sire. The use of a monoclonal a-IX recognizing an exonic polymorphism will be extended from IX to VIII and von Willebrand factor to develop rapid immunoassays for carrier testing in hemophilia A and von Willebrand disease. Attempts will be made to prepare antibodies specific for factor IXa. Peptides to sequences in the variable portion of IXa's heavy chain will be studied and new reagents developed to probe the function of the active enzyme. Binding antibodies in patients with severe defects will be characterized to develop strategies for localization of their defects. A heavy chain defect due to a point mutation in a Taq1 cleavage site will be defined by oligonucleotide probes, to compare with functional data of the hemophilic mutation.